Apparatus and method for ex vivo lung ventilation with a varying exterior pressure

ABSTRACT

In a method of ventilating excised lungs, a ventilation gas is supplied to an airway of a lung and a vacuum is formed around the lung. A quality of the vacuum is varied between a lower level and a higher level to cause the lung to breathe, while the pressure of the ventilation gas supplied to the airway is regulated to maintain a positive airway pressure in the airway of the lung. The vacuum may be cyclically varied between the two vacuum levels. The levels may be maintained substantially constant over a period of time, or one or both of the lower and higher levels may be adjusted during ventilation. The lung may be placed in a sealed chamber, and a vacuum is formed in the chamber around the lung.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 16/305,648, filed Nov. 29, 2018, which applicationis a U.S. National Phase Application under 35 U.S.C. § 371 ofInternational Application No. PCT/CA2017/050643, filed May 26, 2017,which claims the benefit of, and priority to U.S. Provisional PatentApplication No. 62/343,076, filed May 30, 2016, the entire contents ofeach of the prior applications are incorporated herein by reference.

FIELD

This disclosure relates generally to devices and methods for lungventilation, and particularly to devices and methods for ventilation ofexcised lungs by varying exterior pressures.

BACKGROUND

To use excised donor lungs for transplantation, the excised lungs mayneed to be perfused and ventilated ex vivo to restore or preserve theirfunctionalities before the transplant procedure can be performed, or toassess or evaluate their quality or suitability for transplantation.

For ex vivo ventilation of excised lungs, the common traditionalmechanical ventilation techniques employ a positive pressure applied tothe tracheobronchial tree. This creates a pressure gradient between thetracheobronchial tree and the alveoli, such that airflow occurs down thepressure gradient into the alveoli.

It has been recognized that excised lungs can also be ventilated by anegative pressure ex vivo. For example, lungs may be ventilatedutilizing a negative pressure (i.e., below atmospheric pressure) aroundthe lungs to allow the lungs to naturally fill with ventilation gas thatis at or near atmospheric pressure. Some authors have suggested thatdifferent strategies might be combined by supplying positive-pressure(above atmospheric pressure) ventilation gas to the lungs and utilizinga negative pressure around the lungs. However, these authors have notdisclosed any specific details of effective strategies for utilizingpositive and negative pressures to ventilate lungs ex vivo.

SUMMARY

An aspect of the present disclosure relates to a method of ventilatingexcised lungs. In this method, a ventilation gas is supplied to anairway of a lung and a vacuum is formed around the lung. A quality ofthe vacuum is varied between a lower level and a higher level to causethe lung to breathe, while the pressure of the ventilation gas suppliedto the airway is regulated to maintain a positive airway pressure in theairway of the lung. The vacuum may be cyclically varied between the twovacuum levels. The levels may be maintained substantially constant overa period of time, or one or both of the lower and higher levels may beadjusted during ventilation. The lung may be placed in a sealed chamber,and a vacuum is formed in the chamber around the lung.

Conveniently, in some embodiments a single pump may be used to applyboth the airway pressure and the vacuum around the lung.

In a modified embodiment, ventilation is effected by varying theexterior pressure (the pressure applied to the exterior surface of thelungs) between a higher pressure above the atmospheric pressure and alower pressure below the atmospheric pressure. In other words, the lungsmay be caused to breathe by varying the exterior pressure between apositive pressure and a vacuum pressure.

Another aspect of the present disclosure relates to a method ofventilating a lung, comprising applying a first pressure (P1) to anairway of the lung, and applying a second pressure (P2) to an exteriorsurface of the lung. The pressure differential, PD=P1-P2, is maintainedpositive and is varied to cause the lung to breathe.

In an embodiment, the airway pressure P1 is maintained higher than theatmospheric pressure, and the exterior pressure P2 is varied between ahigher pressure level and a lower pressure level, where the lowerpressure level is below the atmospheric pressure. In a particularembodiment, P1 may be maintained at a constant value, such as at aconstant value from about 5 to about 10 cmH₂O. The pressure differentialPD may be varied from about 7 to about 30 cmH₂O. For example, when P1 isconstant at 5 cmH₂O, P2 may vary from −25 to −2 cmH₂O. When P1 isconstant at 10 cmH₂O, P2 may vary from −20 to 3 cmH₂O.

In an embodiment, a regenerative vacuum pump, such as a regenerativeturbine, may be used to apply and control both P1 and P2. P1 may beregulated using the exhaust pressure at the exhaust side of the pump,and P2 may be regulated using the vacuum pressure at the vacuum (intake)side of the pump. Conveniently, a single turbine may be sufficient toapply and control both P1 and P2.

A further aspect of the present disclosure relates to a method ofventilating a lung comprising applying an exterior pressure around alung with a gas in fluid communication with a gas pump, and operatingthe gas pump to vary the exterior pressure around the lung to ventilatethe lung. The gas pump may be a regenerative pump, such as a turbinepump. The gas around the lung may be confined in a constant volume butthe amount of gas (e.g., moles of gas) in the constant volume is variedusing the pump to change the exterior pressure applied to the lung.

In another aspect, a system for ventilating a lung is disclosed, whichcomprises a gas pump comprising an exhaust side and an intake side; asealed chamber for enclosing the lung therein, and configured to apply afirst pressure (P1) to an airway of the lung and apply a second pressure(P2) to an exterior surface of the lung enclosed therein; and aplurality of conduits and a plurality of valves, the conduits and valvesconnecting the gas pump to the sealed chamber for selectively regulatingP1 and P2, wherein the proportional valves comprise a first proportionalvalve connected to the exhaust side of the gas pump, a secondproportional valve connected to the intake side of the gas pump, and athird proportional valve connected to the first proportional valve, andwherein the conduits comprise a first conduit extending through thesealed chamber and connected to the third proportional valve, forconnecting an airway of the lung to the gas pump through the thirdproportional valve and the first proportional valve to supply thepressure applied to the airway of the lung, a second conduit connectingthe second proportional valve to the sealed chamber for supplying thepressure applied to the exterior surface of the lung, and a thirdconduit connecting the second conduit to the first proportional valve,such that P1 is regulated by the first proportional valve and the thirdproportional valve, P2 is regulated by the first proportional valve andthe second proportional valve, and P1 and P2 are independentlyregulatable by controlling the first, second and third proportionalvalves.

In a further aspect, there is provided a lung ventilator comprising acontainer comprising a sealable chamber for housing a lung therein; apump comprising an intake side and an exhaust side; a plurality ofconduits connecting the pump to the sealable chamber for applying afirst pressure (P1) to an airway of the lung and a second pressure (P2)to an exterior surface of the lung; and a control system comprising acontroller, pressure sensors, flow sensors, and flow regulating valves,for controlling operation of the pump and regulating the pressuresapplied to the airway and the exterior surface of the lung, wherein theflow regulating valves comprise a plurality of proportional valvesconfigured to regulate P1 using an exhaust pressure at the exhaust sideof the pump, to regulate P2 using both the exhaust pressure and anintake pressure at the intake side of the pump, such that P1 and P2 areindependently controllable by adjusting proportional valves.

Other aspects, features, and embodiments of the present disclosure willbecome apparent to those of ordinary skill in the art upon review of thefollowing description of specific embodiments in conjunction with theaccompanying figures.

BRIEF DESCRIPTION OF DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present disclosure,

FIG. 1 is a schematic block diagram of an example apparatus forventilating lungs;

FIG. 2 is a schematic block diagram of an example implementation of theapparatus of FIG. 1 ;

FIG. 3 is a schematic block diagram of another example apparatus forventilating lungs;

FIG. 4 is a schematic diagram of the apparatus of FIG. 3 , showing airflow during inspiration;

FIG. 5 is a schematic diagram of the apparatus of FIG. 3 , showing airflow during expiration;

FIG. 6 is a schematic diagram illustrating the control logic used tocontrol the proportional valves in the apparatus of FIG. 3 ;

FIG. 7 is a block diagram of a computer for use with the apparatus ofFIG. 1 or 3 ;

FIG. 8 is a flow chart illustrating an algorithm executed by thecomputer device of FIG. 7 for controlling operation of the apparatus ofFIG. 3 ;

FIGS. 9A and 9B are schematic diagrams of an alveolar network in thelung parenchyma;

FIG. 10 is a line graph illustrating representative intrathoracicpressure (ITP), airway pressure (P_(aw)), and transpulmonary gradient(TPG) profiles over a time period of 4 seconds obtained from sampleporcine lungs ventilated according to an embodiment of the presentdisclosure;

FIG. 11 is a line graph illustrating a pressure-volume profile from thesame experiment as FIG. 10 ;

FIG. 12 is a line graph illustrating representative intrathoracicpressure (ITP), airway pressure (P_(aw)), and TPG profiles over a timeperiod of 4 seconds obtained from sample porcine lungs ventilatedaccording to an embodiment of the present disclosure;

FIG. 13 is a line graph illustrating a pressure-volume profile from thesame experiment as FIG. 12 ;

FIG. 14A shows a line graph (top) in FIG. 14A(a) illustrating arepresentative intrathoracic pressure (ITP) profile over time, with asection of which shown in expanded time scale at the bottom in FIG.14A(b);

FIG. 14B is a line graph of the measured airway pressure over timeduring the same experiment as in FIG. 14A;

FIG. 14C is a line graph of the transpulmonary gradient over time duringthe same experiment as in FIG. 14A;

FIG. 15 is a line graph illustrating a representative lung complianceprofile over time obtained from sample porcine lungs ventilatedaccording to an embodiment of the present disclosure;

FIG. 16 is a line graph illustrating a representative pulmonary vascularresistance (PVR) profile over time obtained from sample porcine lungsventilated with example ventilation apparatus and methods as disclosedherein;

FIG. 17 is a bar graph illustrating edema formation during ex vivo lungperfusion (EVLP) of sample porcine lungs, comparing a combined negativeand positive pressure ventilation strategy (NPV/PPV, using exampleventilation apparatus and methods as disclosed herein) and a typicalpositive pressure ventilation strategy (PPV) with respect to threedifferent perfusates: an acellular mixture, a mixture of whole blood andsolution, and a mixture of red blood cell concentrate (pRBC) andsolution;

FIGS. 18 and 19 are line graphs illustrating representative flow-volumeprofiles obtained from sample human lungs ventilated with exampleventilation apparatus and methods as disclosed herein;

FIGS. 20 and 21 are line graphs illustrating representativepressure-volume profiles obtained from sample human lungs ventilatedwith example ventilation apparatus and methods as disclosed herein; and

FIG. 22 is a schematic diagram of a comparison apparatus for negativepressure ventilation of lungs ex vivo;

FIG. 23 is a line graph illustrating a representative intrathoracicpressure (ITP) profile over time obtained from the container housingsample porcine lungs ventilated with negative pressure according to anapparatus as depicted in FIG. 22 ;

FIG. 24 is a line graph illustrating the step position of the rollerpump over time during negative pressure ventilation of sample porcinelungs using the apparatus depicted in FIG. 22 ;

FIGS. 25A, 25B, and 25C are bar graphs illustrating representativeresults of measurements of lung oxygenation of perfused sample lungsover time, with combined NPV/PPV or PPV;

FIGS. 26A, 26B, and 26C are line graphs illustrating representativeresults of measurements of mean pulmonary arterial pressure of perfusedsample lungs over time, with combined NPV/PPV or PPV;

FIGS. 27A, 27B, and 27C are line graphs illustrating representativeresults of measurements of pulmonary vascular resistance of perfusedsample lungs over time, with combined NPV/PPV or PPV;

FIGS. 28A, 28B, and 28C are line graphs illustrating results ofmeasurements of peak airway pressure of perfused sample lungs over time,with combined NPV/PPV or with PPV;

FIGS. 29A, 29B, and 29C are line graphs illustrating representativeresults of measurements of dynamic compliance of perfused sample lungsover time, with combined NPV/PPV or PPV;

FIGS. 30A, 30B, 30C, 30D, 30E, and 30F are line graphs illustratingrepresentative results of measurements of inflammatory cytokine ofperfused sample porcine lungs over time, with combined NPV/PPV or PPV;and

FIGS. 31A, 31B, and 31C are line graphs illustrating representativeresults of measurements of inflammatory cytokine of perfused samplehuman lungs over time, with combined NPV/PPV or PPV.

DETAILED DESCRIPTION

It has been recognized that, when excised lungs are ventilated ex vivoby varying a pressure around the lungs to cause the lungs to breathe,the lungs may still benefit from application of a regulated positivepressure into the airway to prevent alveolar collapse during expiration.For example, application of a positive airway pressure combined withoscillation of a pressure around the exterior of the lungs to drivebreathing may allow the transpulmonary pressure gradient (TPG) in thelungs to be conveniently regulated to allow for effective recruitment oflung parenchymal alveolar segments, while reducing, minimizing or evenpreventing over distension of recruited segments.

Accordingly, an embodiment of the present disclosure relates to a methodof ventilating excised lungs. In this method, a ventilation gas issupplied to an airway (e.g., the trachea or a bronchus) of a lung and avacuum is formed around the lung. A quality of the vacuum is varied(e.g., cycled) between a lower level and a higher level to cause thelung to breathe, while the pressure of the ventilation gas supplied tothe airway is regulated to maintain a positive airway pressure in theairway of the lung, where the airway pressure may be constant orcontinuously positive. Typically, the vacuum may be cyclically variedbetween the two vacuum levels. The levels may be maintainedsubstantially constant over a period of time, or one or both of thelower and higher levels may be adjusted during ventilation. Theventilation gas may be filtered with a microbe filter and ahumidity-moisture-exchanger (HME) filter before being supplied into thelung. The lung may be placed in a sealed chamber, and a vacuum is formedin the chamber around the lung.

An example apparatus 10 for ventilating excised lungs is schematicallyillustrated in FIG. 1 .

As depicted, the apparatus 10 includes a container 12 having a sealablechamber 14 for housing a lung 50. For clarity, it is noted that the term“a lung” can refer to a single lung, multiple lungs, or a portion of asingle lung or lungs. Two lungs attached to the same trachea aresometimes collectively referred to as “a lung” or “the lung” herein.

A ventilation gas source 16 is provided for supplying a ventilation gasat a variable positive pressure.

As used herein, a positive pressure refers to a pressure that is higherthan the atmospheric pressure in the immediate environment of lung andthe ventilation device, unless otherwise specified expressly.

A first conduit 18 extends through the wall of the container 12 andconnects the ventilation gas source 16 to an airway 52 of the lung 50,for supplying the ventilation gas to the airway of the lung. The conduit18 is sealed from pressure communication with the inner space in thechamber 14. As will be further described below, the ventilation gas maybe air or any suitable gas mixture that contains oxygen. The ventilationgas source 16 may include the output port of an air pump or amotor-driven turbine (not shown in FIG. 1 , but see FIG. 2 ) forsupplying air to the lung at a positive pressure. The operation speed ofthe air pump or turbine may be controlled to regulate airway pressure inthe lung.

A second conduit 20 connects a vacuum source 22 to the chamber 14 forforming a vacuum in the chamber 14. The same turbine used to supply theventilation gas may be used to provide the vacuum source 22 (see e.g.,FIG. 2 ).

A control system 24 is coupled to the ventilation gas source 16 and thevacuum source 22. As will be further described in more detail below, thecontrol system 24 may include pressure sensors, flow sensors,flow-regulating valves, and one or more controllers (not shown in FIG. 1, but see FIG. 2 ), which are configured and adapted to vary a qualityof the vacuum in chamber 14 between a lower vacuum level and a highervacuum level to cause the lung 50 to breathe, and to regulate thepressure of the ventilation gas supplied by the ventilation gas source16 to maintain a continuously positive airway pressure in the airway 52of the lung 50.

For example, as illustrated in FIG. 2 , which illustrates an exampleimplementation 10A of the apparatus 10, a motor-driven turbine 30 may beused as the ventilation gas source 16 to supply air to the lung 50 fromthe output port of the turbine 30, and used as the vacuum source 22 toform and vary the vacuum in the chamber 14 by drawing or sucking airfrom the chamber 14 into the intake port of the turbine 30. In theexample implementation 10A, the pressure in chamber 14 can also bepositive (i.e., above atmospheric pressure) at selected times.

The control system 24 may include a first proportional valve 32 coupledto the conduit 18 for selectively releasing a portion of the air in theconduit 18 to the atmosphere (as indicated by the arrow above valve 32in FIG. 2 ), and a second proportional valve 34 coupled to the conduit20 for selectively adding air from the atmosphere into the conduit 20(as indicated by the arrow above valve 34 in FIG. 2 ). As can beappreciated, in a different embodiment if a different ventilation gassource may be used to replace air in the atmosphere.

The control system 24 may also include a firstproportional-integral-derivative (PID) controller 36 for controlling theoperation of the first proportional valve 32, and a second PIDcontroller 38 for controlling the operation of the second proportionalvalve 34.

A third conduit 40 may be provided to interconnect conduits 18 and 20,and a third proportional valve 42 may be connected to the output port ofthe turbine 30 and coupled to the conduits 18 and 40 for selectivelyfeeding air from the output port of the turbine 30 into the conduits 18and 40 in different proportions. The second PID controller 38 may alsobe connected to control the operation of the third proportional valve42.

The control system 24 may further include a first pressure sensor 44 forsensing a first pressure in the first conduit 18, and a second pressuresensor 46 for sensing a second pressure in the chamber 14. The firstpressure sensor 44 is connected to provide an input to the first PIDcontroller 36, and the second pressure sensor 46 is connected to providean input to the second PID controller 38.

The control system 24 may further include a central processing unit or acontroller 48 in electrical communication with the PID controllers 36and 38 and the turbine 30, for controlling the operation of the PIDcontrollers 36 and 38 and the operation of the turbine 30. Thecontroller 48 may be a microprocessor, and may be provided in the formof a computer (see e.g., FIG. 7 ).

While not shown in FIGS. 1 and 2 , one or more microbe and HME filters(see e.g., FIG. 3 ) may be coupled to one or more of conduits 18 and 20for filtering and humidifying the air to be supplied into the chamber 14and the airway 52 of the lung 50.

Embodiments of the method and apparatus described herein may beconveniently used for negative pressure ventilation in an ex vivo lungperfusion (EVLP) process or system. Application of positive pressureinto the airway of the lung, when combined with such negative pressureventilation, allows a higher TPG to be achieved without applyingexcessively negative pressure to the exterior of the lung.

Embodiments disclosed herein may also allow for recovery of atelectaticalveoli, thereby facilitating extended EVLP. It is further convenient touse at least some disclosed embodiments to measure and obtain functionalattributes of the ventilated lungs ex vivo.

A single suitable turbine can generate a sufficient pressure gradientand airflow to meet the requirements for ventilating lungs with avariety of sizes. A single turbine can provide both a source of vacuum(e.g., for applying a negative pressure to the exterior of a lung) and asource of positive pressure (e.g., for applying a positive pressure tothe airway of the same lung, and optionally applying a positive pressureto the exterior of the lung). Thus, in an embodiment disclosed herein asingle turbine may be sufficient to drive the air flows in theventilation system. A benefit of such an embodiment is that it is energyefficient, as the same energy used to generate the vacuum is also usedto generate the positive pressure. Another benefit of such an embodimentis its relative simple construction and small footprint.

In a further embodiment, a combined ventilation and perfusion apparatus100 may be constructed as illustrated in FIG. 3 .

As depicted, donor lungs 150 are placed inside a rigid orpressure-resistant container 110. Within the container 110, the lung maybe supported on a flexible porous surface, such as a silicone or plasticnet, or the lung may be rendered buoyant through placement on a fluidsurface covered with a soft plastic membrane (not shown). Alternatively,the lung may be supported on a semi-rigid plastic form that resemblesthe shape of the posterior chest such that the lungs lie in ananatomically familiar position (not shown).

A perfusion apparatus 160 is provided to perfuse the lungs 150. Aconduit 162 connected to the perfusion apparatus 160 is also connected,optionally with a cannula, to a pulmonary artery 154 of the lungs 150. Aconduit 164 connected to the perfusion apparatus 160 is also connectedwith a pulmonary vein 156, possibly through attachment to the leftatrium and optionally with a cannula, of the lungs 150. Through conduits162 and 164, the perfusion apparatus 160 can be configured to circulatea perfusate through the vasculature of the lungs 150 in a manner knownto those skilled in the art.

The tracheobronchial tree 152 of the lungs 150 is connected to a conduit142 by an endotracheal tube 144. As will be apparent to a person ofskill in the art, when a single lung or a portion of a single lung ismounted in the apparatus, an endotracheal tube or analogous device canbe inserted into either the trachea attached to the lung (or lungportion) or inserted directly into a bronchus of the lung (or lungportion). In such instances, a pediatric endotracheal tube may be theappropriate size to connect to a bronchus.

The container 110 is sealed with a lid 112 to isolate the inner space incontainer 110 from the atmosphere. The conduit 142 (or the endotrachealtube 144) passes through the lid 112 via a port 114. The conduit 162passes through the lid 112 via a port 116. The conduit 164 passesthrough the lid 112 via a port 118. When the conduits 162, 164 and 142(or the endotracheal tube 144) are installed in place, all of the ports114, 116 and 118 are sealed to the atmosphere. As a result, the innerspace in the container 110 is isolated from the atmosphere, and thepressure exerted on the exterior surfaces of the lungs 150 is notdependent on the atmospheric pressure and can be independentlycontrolled and regulated.

A conduit 136 connects the container 110 to a conduit 134. The gaspressure inside the container 110 is dependent on the pressure insidethe conduit 136, and the pressure in the conduit 134. The conduit 134 isconnected to proportioning valves 104 and 106, which are in turnconnected to the intake port and output port of a turbine 102 byconduits 130 and 132 respectively.

The valve 104 has an open inlet 138 that allows atmospheric air to enterthe valve 104, and can be operated to allow selected proportions of airfrom the atmosphere (“atm”) and the conduit 134 to enter the conduit130.

The valve 106 is coupled to the conduits 132, 134 and 140, and isoperable to allow selected proportions of air from the conduit 132 toenter either the conduit 134 or the conduit 140.

As can be appreciated, the turbine 102 outputs a positive pressure atthe output port connected to the conduit 132, and forms a negativepressure at the intake port connected to the conduit 130.

The atmospheric air may be filtered for microbes and other particlesbefore passage through the valve 104 by a filter (not shown).

Optionally, a source of a gas mixture (e.g., oxygen/air) at-or-aroundatmospheric pressure may be connected to inlet 138 of the valve 104, tosupply a ventilation gas in place of atmospheric air, so as to exposethe lungs to a desired or controlled gas mixture.

In the example apparatus in FIG. 3 , the rotational speed of the turbine102 may be varied to control the air pressure applied at the output portof the turbine 102, although it is also possible to operate turbine 102at a constant speed over a period of time if desired. As can beappreciated, when turbine 102 is in normal operation the air pressureinside the conduit 130 is a negative pressure (i.e., lower than theatmospheric pressure), and the air pressure inside the conduit 132 is apositive pressure (i.e., higher than the atmospheric pressure).

The valves 104 and 106 can be controlled, such as by a controller 170particularly a subcontroller 172 in the controller 170, to regulate thepressure in the conduits 134 and 136, and consequently the pressure inthe inner space of the container 110 to form a vacuum in the container110 around the lungs 150. The valves 104 and 106 can be controlled tooscillate the pressure inside the container 110 between a lower (vacuum)pressure and a higher (vacuum or positive) pressure, which will causethe lungs to breathe (i.e., taking in and expelling out air through theendotracheal tube 144). It should be understood that the term “vacuum”as used herein refers to partial vacuum, and the quality of the vacuumrefers to how closely the vacuum approaches a perfect vacuum. In otherwords, the quality of the vacuum is related to the vacuum pressure, andhow close the vacuum pressure approaches absolute zero. The variation inthe air pressure in the container 110 causes the lungs 150 tocorrespondingly expand or contract. The lungs may contract even when theairway pressure in the endotracheal tube 144 is higher than the instantair pressure in the container 110, as long as the pressure differentialis not too high so that the pressure differential can be overcome by theelastic recoil of the lungs. Expansion and contraction of the lungs 150can be controlled to mimic or simulate the expansion and contraction ofin vivo lungs during normal breathing, and to move air into and out ofthe alveoli through the endotracheal tube 144. With the controllers asdescribed, the apparatus 100 allows precise control and regulation ofthe pressures and the rates of pressure change in both the container 110and in the endotracheal tube 144, and the waveforms or profile of thepressure oscillation can be conveniently set and varied by a user, suchas using a computer 200.

The turbine 102 can be used to generate a basal level of airflow throughthe system, which generates a pressure gradient. The pressure gradientbetween the tracheobronchial tree and the serosal surface of the lungsmay be varied by adjusting the proportioning valves 104, 106 and 108 tovary the TPG such that the lungs cyclically inhale and exhale. Theturbine speed may be varied only when needed to ensure the pressuregradient is sufficient throughout the each ventilation cycle.

Although not necessary for negative pressure ventilation, as notedabove, maintaining a positive airway pressure in the endotracheal tube144 can provide certain benefits and advantages. In this regard, theconduit 140 is connected to the conduit 142 through a thirdproportioning valve 108, which has an open outlet 146. The valve 108 isoperable to supply a selected portion of air from the conduit 140 intothe conduit 142, and the remaining portion of air is released into theatmosphere (atm) through the outlet 146. As can be appreciated, indifferent embodiments when a ventilation gas other than atmospheric airis used, outlet 146 may be connected to the source of the ventilationgas to recycle or circulate the ventilation gas back to the source.

As one example, positive airway pressure can be achieved during ex vivoventilation by applying a continuous or constant positive pressure intothe airway, such as in a similar manner as the airway pressures appliedin a treatment technique known as continuous positive airway pressure(“CPAP”) in the treatment of some human disorders (e.g., obstructivesleep apnea). For clarity, the terms “continuous” and “continuously” asused herein are not synonymous with the term “constant”.

The valve 108 is controlled by the controller 170, particularly thesubcontroller 174 in the controller 170, to regulate the pressure andflow rate in the conduit 142, and consequently the airway pressure inendotracheal tube 144. The valve 108 may also allow air in the conduit142 be released into the atmosphere when the lungs 150 are caused by thehigher pressure in the container 110 to expel air from the lungs 150.The valve 108 may be controlled to maintain the desired airway pressure,where the desired pressure may be set by the user to be in the range ofatmospheric pressure up to an upper limit (e.g., 10 cmH₂O).

To avoid desiccation of the lungs 150, a HME filter 124 can be coupledto the conduit 136, and a HME filter 126 can be coupled to the conduit142. Optionally, microbe filters can also be coupled to the conduits 136and 142 (not shown).

As alluded to earlier, the operation of the valves 104, 106, and 108 iscontrolled by a controller 170, based on signals received from apressure sensor 122 coupled to the conduit 142, which detects a pressuresignal that is indicative of the airway pressure (P_(AW)) in the lungs150 and a pressure sensor 120 coupled to the container 110 for detectinga signal indicative of the pressure in the container 110 (referred to asthe “intrathoracic” pressure or P_(IT) or ITP). One form the controller170 may take is a computer 200 (not shown in FIG. 3 ).

The controller 170 may also be connected to a flow sensor 128 thatdetects a signal indicative of the air flow rate in the endotrachealtube 144. Optionally, the valves 104, 106, and 108 may be operated basedin part on the signal received from the flow sensor 128.

The rotational speed of the turbine 102 may be controlled by thecontroller 170 or the computer 200 based on the detected signals and oneor more parameters set by a user.

The user set-points for the controller 170 or the computer 200 mayinclude the end inspiratory pressure (EIP) in the container 110, the endexpiratory pressure (EEP) in the container 110, the inspiratory time(T_(i)), the expiratory time (T_(e)), the tidal volume (V_(t)), and theairway pressure (P_(AW)). As will be apparent to a person of skill, whenventilation is effected by varying an exterior pressure around thelungs, EIP and EEP refer to the pressure levels of the exterior pressure(e.g., the pressure inside container 14 in FIGS. 1-2 or container 110 inFIGS. 3-5 ), which pressure is also referred to herein as the“intrathoracic” pressure (abbreviated as P_(IT) or ITP). By contrast, intraditional mechanical ventilation techniques in which a varyingpositive pressure is applied into the airway to cause ventilation, EIPand EEP are usually measures of the levels of the airway pressure atdifferent points in a ventilation cycle.

The controller 170 or the computer 200 may use intrathoracic airpressure (P_(IT)), airway pressure (P_(AW)) and endotracheal tubeairflow (V) as inputs. The controller 170 or the computer 200 may outputcontrol signals for controlling the three proportional valves 104, 106,and 108 and the motor or turbine speed (e.g., in terms of rotations perminute) of the turbine 102.

As can be appreciated, the controller 170 and the computer 200 mayreceive additional inputs from other components shown or not shown inthe figures, and may be used to control additional components of theapparatus 100. For example, a temperature sensor (not shown) and atemperature control device (not shown) may be used and connected to thecomputer 200 or the controller 170 to control the temperature incontainer 110. In addition, the computer 200 or the controller 170 maybe used to control the flow of perfusate through the pulmonaryvasculature.

The dotted or dashed lines in the figures (such as FIGS. 1, 2 and 3 )indicate communication connections, which may be electrical orotherwise, and may be wired connections or wireless connections as canbe understood by those skilled in the art.

The controller 170 may include one or moreproportional—integral—derivative (PID) controllers, although two PIDsubcontrollers 172 and 174 are depicted in FIG. 3 .

In the particular example embodiment depicted in FIG. 3 , the PIDsubcontroller 172 in the controller 170 uses the pressure in thecontainer 110 (detected by the pressure sensor 120) as an input (I_(a)),and outputs a signal (O_(b), O_(c)) for controlling the proportionalvalves 104 and 106. The PID subcontroller 174 in the controller 170 usesthe airway pressure (I_(x)) detected by the pressure sensor 122 (andoptionally the endotracheal air flow rate (I_(y)) measured by the flowsensor 128) as an input, and outputs a signal (O_(z)) for controllingthe proportional valve 108.

The turbine 102 is optionally connected to and controlled by thecomputer 200 or the controller 170.

The controller 170 may be configured by a user with differentuser-selected parameter settings or different series of parametersettings (e.g., desired container pressures over time), which may beentered by a user into the controller 170 using a user interface such asa graphical user interface (GUI) (not shown), or may be loaded from aconfiguration file stored in a computer memory. The parameter settingsmay include set-point values for one or more PIDs in the controller 170.

For example, a positive airway pressure may be maintained in thetracheobronchial tree 152 by properly setting the parameter for the PIDsubcontroller 174 set-point values to control the endotracheal tube flowand airway pressure. These set-point values may or may not change overtime.

Conveniently, an apparatus disclosed herein such as apparatus 100 canalso be used to measure and store functional attributes of the lungs150, such as the flow-volume profile or pressure-volume profile for apair of ventilated lungs. The volume may be measured or calculated basedon airflow as detected by flow sensor 128. Other functional attributesthat can be measured with the apparatuses and methods of the disclosureinclude dynamic compliance, elastance, and airway resistance.

Examples of suitable turbines include a turbine used in the PhilipsRespironics Duet LX™ CPAP Pro machines, and may include other knownturbines that are suitable for use in conventional CPAP treatment ofintact lungs (such as those disclosed in EP 1488743 published Dec. 22,2004 or U.S. Pat. No. 6,526,974 to Brydon et al. published Mar. 4,2003).

Other example turbines are described at the following URLs:

-   -   http://mag.ebmpapst.com/en/industries/medical/the-secret-of-the-turtle_2433/    -   https://www.bedek.de/en/blowers-and-fans-in-medical-filed.html    -   http://www.micronel.com/products/micronel-inside-medical/

Suitable examples of proportioning valves include those used in Philips™Respironics BiPAP machines, and may include those with a voicecoilactuator.

Any suitable microbe filters, such as high-efficiency particulatearresting (HEPA) filters, and HME filters known to those skilled in theart may be used in an embodiment herein. There are products on themarket that have both HME and HEPA properties.

FIG. 4 illustrates the air flow in apparatus 100 during inspiration. Thearrows alongside conduits indicate the direction of airflow. The valvesare configured such that air flows from inside the container 110, intothe conduit 136, and then into the conduit 134 before moving through theturbine 102. In this manner, the pressure inside the container 110 isdecreased and negative pressure is applied to the exterior of the lungs150. The air flows from the turbine outlet, into the conduit 132,through the valve 106, into the conduit 140, through the valve 108, andinto the conduit 142 and the endotracheal tube 144. In this manner, apositive pressure is applied to the airway 152 of the lungs 150. Thecombination of negative and positive pressure results in a pressuregradient from the tracheobronchial tree to the alveoli, such thatairflow occurs down the pressure gradient into the alveoli.

FIG. 5 illustrates the air flow in the apparatus 100 during expiration.The valves are configured such that air flows from the inlet 138,through the valve 104, into the conduit 130, through the turbine 102,and into the conduit 132. As the valve 106 is open, at an appropriateproportion, to both the conduit 134 and the conduit 140, air flows intothe chamber 110 and toward the valve 108. Increasing the pressure insidethe chamber 110, when combined with the elastic recoil of the lungs,results in a pressure gradient from the alveoli to the tracheobronchialtree, such that airflow occurs down the pressure gradient into thetracheobronchial tree 152, out of the endotracheal tube 144, into theconduit 142, and then into the proportioning valve 108, from which theexpired air exits the apparatus through the outlet 146. The valve 108appropriately proportions the airflow from the conduit 140 into theconduit 142 and the outlet 146 such that the positive pressure into theendotracheal tube 144 does not impede expiration.

FIG. 6 illustrates the control logic for controlling the valves 104,106, and 108. The control may be implemented using aproportional-integral-derivative (PID) controller, although the PIDcontroller may be used to provide P-I control, P-D control, P control orI control. As can be understood by persons skilled in the art, a PIDcontroller can continuously calculate an error value as the differencebetween a desired set-point and a measured variable or multiple detectedvariables. A PID controller can attempt to minimize the error value or acomposite of multiple error values over time by adjustment of acontrolled variable. The set-points are entered by the user, eithermanually or by loading set-points from a memory device. The top PIcontrol logic in FIG. 6 , which is provided by subcontroller 172 in FIG.3 , is used to control proportional openings of valves 104 and 106. Inthis logic, the detected pressure inside the container 110 housing thelungs 150 (the “intrathoracic pressure” or P_(IT)) is compared to aset-point of the desired P_(IT), and the difference between the actualP_(IT) and the set point is used as feedback for adjusting the valve 104and the valve 106. The bottom PI control logic, which is provided bysubcontroller 174 in FIG. 3 , is used to control proportional opening ofvalve 108. In this logic, the detected pressure inside the airway of thelungs (P_(AW)) and the measured or calculated endotracheal tube airflow(V) are compared to their respective set-points, and the respectiveerrors are used for adjusting the proportioning in the valve 108.

FIG. 7 is a high-level block diagram of the computing device 200, whichcan be used in combination with other controllers or in place of thecontroller 170. The computing device 200 may include or be part of aportable computing device (e.g., a mobile phone, netbook, laptop,personal data assistant (PDA), or tablet device) or a stationarycomputer (e.g., a desktop computer, or set-top box). As will becomeapparent, the computing device 200 includes software that allows a userto control and monitor an ex vivo lung ventilation apparatus, such asthe apparatus 100 in FIG. 3 . As illustrated, the computing device 200includes one or more processors 202, a memory 206, a network interface208 and one or more I/O interfaces 204 in communication over a bus 210.One or more processors 202 may be one or more INTEL™ x86, INTEL™ x64,AMD™ x86-64, POWERPC™, ARM™ processors or the like. The memory 206 mayinclude random-access memory, read-only memory, or persistent storagesuch as a hard disk, a solid-state drive or the like. Read-only memoryor persistent storage is a computer-readable medium. A computer-readablemedium may be organized using a file system, controlled and administeredby an operating system governing overall operation of the computingdevice. The network interface 208 serves as a communication device tointerconnect the computing device 200 with one or more computer networkssuch as, for example, a local area network (LAN) or the Internet. Thenetwork interface 208 may be configured to enable the computing device200 to communicate with external devices via one or more networks. Thenetwork interface 208 may be a network interface card, such as anEthernet card, an optical transceiver, a radio frequency transceiver, orany other type of device that can send and receive information. One ormore I/O interfaces 204 may serve to interconnect the computing device200 with peripheral devices, such as, for example, keyboards, mice,video displays, and the like (not shown). Optionally, the networkinterface 208 may be accessed via one or more I/O interfaces 204. One ormore I/O interfaces 204 may serve to collect information from andcontrol components of the apparatus of the disclosure, of which theapparatus 100 in FIG. 3 is an example. For instance, an I/O interface204 may communicate by wire or wirelessly with valves, pressure sensors,a flow sensor, and a turbine. The I/O interfaces 204 may be configuredto receive input from a user. Input from a user may be generated as partof a user running one or more software applications.

Software comprising instructions is executed by one or more processors202 from a computer-readable medium. For example, software may be loadedinto random-access memory from persistent storage of the memory 206 orfrom one or more devices via I/O interfaces 204 for execution by one ormore processors 202. As another example, software may be loaded andexecuted by one or more processors 202 directly from read-only memory.

The memory 206 stores an operating system 212, applications 214, and aventilation application 216. The operating system 212 may be configuredto facilitate the interaction of applications, such as an application214 and a ventilation application 216, with processor(s) 202, memory206, I/O interfaces 204, and the network interface 208 of the computingdevice 200.

The operating system 212 may be an operating system designed to beinstalled on laptops and desktops. For example, the operating system 212may be a Windows operating system, Linux, or Mac OS. In another example,if the computing device 200 is a mobile device, such as a smartphone ora tablet, the operating system 212 may be one of Android, iOS or aWindows mobile operating system.

The applications 214 may be any applications implemented within orexecuted by the computing device 200 and may be implemented or containedwithin, operable by, executed by, and/or be operatively/communicativelycoupled to components of the computing device 200. The applications 214may include instructions that may cause the processor(s) 202 of thecomputing device 200 to perform particular functions. The applications214 may include algorithms which are expressed in computer programmingstatements, such as, for loops, while-loops, if-statements, do-loops,etc. Applications may be developed using a programming language.Examples of programming languages include Hypertext Markup Language(HTML), Dynamic HTML, Extensible Markup Language (XML), ExtensibleStylesheet Language (XSL), Document Style Semantics and SpecificationLanguage (DSSSL), Cascading Style Sheets (CSS), Synchronized MultimediaIntegration Language (SMIL), Wireless Markup Language (WML), Java™,Jini™, C, C++, Perl, Python, UNIX Shell, Visual Basic or Visual BasicScript, Virtual Reality Markup Language (VRML), ColdFusion™ and othercompilers, assemblers, and interpreters.

The ventilation application 216 is an example of an applicationconfigured to ventilate lungs ex vivo according to the techniquesdescribed herein. As described above, the controller 170 or thecomputing device 200 may include GUIs that enable a user to monitorand/or control one or more ventilation parameters (e.g., P_(IT)). Theventilation application 216 may be configured to enable a user tomonitor and/or control ventilation parameters using one or more GUIs.

It should be noted that although the example computing device 200 isillustrated as having distinct functional blocks, such an illustrationis for descriptive purposes and does not limit the computing device 200to a particular hardware architecture. Functions of the computing device200 may be realized using any combination of hardware, firmware and/orsoftware implementations.

FIG. 8 is a flow chart of an algorithm S300 that can be executed by oneor more processors 202 to monitor and control an apparatus of thedisclosure, such as the apparatus 100 in FIG. 3 .

The software is initiated by a signal provided by a user or automatedprocess at block S302. At block S304, one or more processors 202 receiveinitial settings to configure the apparatus 100.

At block S306, one or more processors 202 communicates with one or moreI/O interfaces 204 to initiate control of the apparatus 100 once thelungs 150 are attached and the container 110 sealed.

At block S308, one or more processors 202 communicates with one or moreI/O interfaces 204 to receive information (e.g., P_(IT), P_(AW), V) fromsensors in the apparatus 100. Some or all of this information from thesensors is displayed at block S310, and the display may be continuallyor periodically updated with information received from the sensors. Atblock S312, the software determines the desired set-points at thatmoment in time, as entered by a user, either manually or by being placedinto the memory 206 (e.g., by loading a configuration file). Theset-points may change with the time in order to cause the pressureoscillations that cause the lung to breathe ex vivo. The software thendetermines at block S314 whether adjustments are required. If so, atblock S316 adjustments are made to components of the apparatus 100(e.g., to the valves 104 and 106 to alter P_(IT)). Block S314 and blockS316 may include PID calculations, as discussed with respect to FIGS. 3and 6 .

At block S318, the status of the apparatus 100 is communicated to theuser by updating the display, and at block S320 the software checks foruser input (e.g., to change set-points). At block S322, it is determinedwhether ventilation is done, either according to predetermined settingsor through live interfacing with a user. If ventilation is not done, thesoftware returns to block S308 to again sample sensors.

If ventilation is done, at block S322, it is determined whether theoperation settings should be reconfigured, such as by loading a newconfiguration file. Reconfiguration may be required when a differentmode of operation is desired. If the settings are to be reconfigured,the software returns to block S304 to receive new initial settings (notshown). If the settings are not to be reconfigured, ventilation isstopped at block S324.

Another aspect of software S300 may be the recording of information fromsensors in the apparatus and adjustments S316 made by the software. Thisinformation may be stored in the memory 206.

An embodiment of the present disclosure can provide improvedperformance.

For example, it is noted that when a positive pressure is applied to theairway in the lung ex vivo and there is a pressure gradient from thetracheobronchial tree to the alveoli, if there is a large discrepancybetween the surface tension of the alveolar group, the high pressure inthe tracheobronchial tree preferentially goes to alveoli with lowerdistending pressure, which may result in over-distension of thosealveoli, whereas alveoli with higher distending pressure are notventilated. This can lead to over-distension of healthy alveoli, withincomplete recovery of atelectatic (collapsed alveoli with high surfacetension) lung segments. As alveoli rupture from high pressure that isgiven in attempts to recover atelectatic alveoli, bullae can form on thesurface of the lung and subsequently rupture causing air leak andfurther injury to the lung.

It is also noted that lungs are naturally ventilated in vivo in the bodythrough an increase in negative pressure to the serosal surface of thelungs. Two layers of serous membrane enclose each lung; the parietalpleura line the wall of the thoracic cavity and the visceral pleuracovers the lung. The pleural cavity between the two pleurae contains asmall amount of lubricating fluid. During normal breathing, the pressurein the pleural cavity, called intrathoracic pressure, is alwayssubatmospheric. During inspiration, muscle contraction increases theoverall size of the thoracic cavity, decreasing intrathoracic pressure.This negative pressure is transmitted throughout the lung parenchyma andalveolar network, which creates a pressure gradient from thetracheobronchial tree to the alveoli. As a consequence, air flows intothe alveoli during inspiration. During normal expiration in vivo, themuscles of inspiration relax. Expiration results from elastic recoil ofthe chest wall and lungs, with much of the inward pull caused by thesurface tension in the film of alveolar fluid. This elastic recoilcreates a pressure gradient from the alveoli to the tracheobronchialtree, such that air flows out of the alveoli. During forceful expirationin vivo, muscles of expiration contract, actively increasingintrathoracic pressure. During a forceful expiration, such as during acough, intrathoracic pressure may briefly exceed atmospheric pressure.

The “pull” effect of negative pressure on the alveoli during inspirationprovides for an equal distribution of the expanding force across theentire population of alveoli. FIGS. 9A and 9B illustrate this effect,with a schematic of the alveolar network in the lung parenchyma.

In FIGS. 9A and 9B, the central circle 502 represents a bronchiolearound an airway, the outer circle 503 represents the exterior surfaceof the lung, and each hexagon 504, 506 or 508 represents an alveolus.The degree of openness of each alveolus is indicated by its shading,such that white shading indicates an open alveolus 504, lined shadingindicates a partially closed alveolus 506, and black shading indicates afully closed alveolus 508. Further, the thickness of the linessurrounding each alveolus in FIGS. 9A and 9B indicate alveolar surfacetension (i.e. distending pressure), such that thicker lines indicatehigher surface tension and thinner lines indicate relatively lowersurface tension. FIG. 9A illustrates that positive pressure directedinto the airway exerts pushing forces (indicated by the arrows 512) thatare unevenly transmitted to the alveoli, such that some alveoli 506 and508 remain partially or fully closed. FIG. 9B illustrates that anegative pressure applied on the exterior surface of the lung exertspulling forces (indicated by the arrows 510) that are evenly transmittedto the alveoli.

In other words, the central circle in each of FIGS. 9A and 9B representsthe wall 502 of an airway (e.g., a bronchiole), the outer circleindicates the exterior surface of a lung 503, and each hexagonrepresents an alveolus, where each alveolus may be open (shown as whitealveoli, e.g., alveoli 504, 505), partially closed (indicated by hashlines, e.g., alveolus 506), or fully closed (shown as black alveoli,e.g., alveolus 508). The alveoli are interconnected (not shown) from thelung surface 503 to the airway. In the intact organism, negativepressure is exerted on the surface of the lung 503 to pull the alveoliopen (FIG. 9B). This pulling force, as indicated by block arrows (e.g.,510), is evenly transmitted throughout the lung parenchyma to theairway, resulting in a population of open alveoli (e.g., 505). Incontrast, with conventional positive pressure ventilation, the airway ispressurized to forcibly fill the alveoli with air (FIG. 9A).Transmission of air pressure, as indicated by block arrows (e.g., 512),will follow the path of least resistance leading to overexpansion ofcompliant alveoli (e.g., 504), leaving less compliant alveoli partiallyunaerated (e.g., 506) or fully unaerated (e.g., 508).

In addition, movement of an alveolus stimulates surfactant productionfrom Type II pneumocytes, which facilitates reduction in the surfacetension inside the alveolus, facilitating its expansion duringinspiration. Without surfactant, the surface tension in an alveolus isvery high, resulting in a very high distending pressure, which hampersairflow.

Even after a forced expiration in vivo, considerable air remains inhealthy lungs because the subatmospheric intrathoracic pressure keepsthe alveoli slightly inflated. However, in patients with certaindisorders, significant alveolar closure occurs at the end of expiration,which decreases lung compliance during the following inspiration.Opening closed alveoli requires a critical pressure to be achievedbefore the alveoli can expand. Such patients can benefit from theapplication of a positive end-expiratory pressure (PEEP) or CPAP whichprevent alveolar closure during expiration.

A more extreme problem with alveolar closure occurs during lungtransplantation. When the lungs are removed from the host body, exposureof the exterior of the lungs to atmospheric pressure can lead towidespread atelectasis.

However, when the lungs are ventilated according to an embodiment of thepresent disclosure, effective recruitment of lung parenchymal alveolarsegments may be obtained, and over distension of recruited segments maybe conveniently reduced or avoided. In particular, a desired pressuregradient in the lungs may be conveniently provided by adjusting thepressure outside the lungs (or pressure in the ventilation container)and the positive pressure in the airways of the lungs.

In an embodiment, a method of ventilating a lung may include applying afirst pressure (P1) to an airway of the lung, and applying a secondpressure (P2) to an exterior surface of the lung. The pressuredifferential, PD=P1−P2, is maintained positive and is varied to causethe lung to breathe. PD may be considered to be equivalent to TPG. Theairway pressure P1 may be maintained higher than the atmosphericpressure, and the exterior pressure P2 may be varied between a higherlevel and a lower level to cause the lung to ventilate, where the lowerlevel is below the atmospheric pressure. P1 may be maintained at aconstant value, such as at a constant value from about 5 to about 10cmH₂O. PD may be varied from about 7 to about 30 cmH₂O. For example,when P1 is constant at 5 cmH₂O, P2 may vary from −25 to −2 cmH₂O. WhenP1 is constant at 10 cmH₂O, P2 may vary from −20 to 3 cmH₂O. Aregenerative vacuum pump, such as a regenerative turbine, may be used toapply and control both P1 and P2. P1 may be regulated using the exhaustpressure at the exhaust side of the pump, and P2 may be regulated usingthe vacuum pressure at the vacuum (intake) side of the pump.Conveniently, a single turbine may be sufficient to apply and controlboth P1 and P2.

In comparison, positive displacement pumps, such as roller pumps orperistaltic pumps, are not as convenient to use to control the airwayand exterior pressures in some embodiments disclosed herein. One of thereasons is that, as will be understood by those skilled in the art, itis more difficult to precisely control the fluid pressures at the inlet(intake) and outlet (output) of a positive displacement pump byadjusting its pumping speed, as compared to a regenerative pump. Forexample, with a positive displacement pump, the inlet pressure (or thepressure differential across the inlet and the outlet of the pump) canvary at the same given pumping speed, so that changing the pumping speedmay not provide a predictable pressure change. With a regenerative pump,the pumping speed is more predictably related to the pressuredifferential across the inlet (intake) and the outlet (output).

Normal physiology also informs the magnitude of the pressures that mayprovide for effective ventilation of lungs ex vivo. Normal physiologydictates P_(AW) and the pressure inside alveoli is 0 cmH₂O (i.e., atatmospheric pressure) at rest, with an intrathoracic EEP around −7 cmH₂Obeing resisted by an elastic recoil of the lungs of approximately thesame magnitude. Deep inspiration can invoke an intrathoracic EIP of −30cmH₂O, whereas a cough or valsalva maneuver can induce an intrathoracicpressure of 100 to 200 cmH₂O. Because of the alveolar network thatapplies traction from the surface of the lungs to the central airway (asillustrated in FIGS. 9A and 9B), application of a negative pressure tothe outside of the lungs is expected to be a physiological method forcausing air movement into the lung. However to reduce the amount ofvacuum applied to lungs being ventilated ex vivo, a small positive airpressure can be applied to the airway to yield a TPG that is maintainedwithin the physiologic range. For example, application of a positivepressure into the airway of between 5 to 10 cmH₂O can reduce therequired vacuum applied to the exterior surface of the lungs by anequivalent amount. An airway pressure above this amount is consideredless physiologic and may be undesirable.

In another embodiment, a method of ventilating a lung includes varyingan exterior pressure around a lung to ventilate the lung. The exteriorpressure is applied by a gas in fluid communication with a gas pump forvarying the exterior pressure. The gas pump may be a regenerative pump,such as a turbine pump. The gas around the lung may be confined within aconstant volume (e.g. between fixed walls) but the amount of gas (e.g.,moles of the gas) in the constant volume is varied using the pump tochange the exterior pressure applied to the lung.

It has been recognized that when a varying pressure is applied to theexterior surface of the lungs using a fixed amount of gas confinedaround the lungs by compressing or decompressing the fixed amount ofgas, such as by varying the volume that the gas occupies, a problem withpressure control could arise. For example, due to air leakage throughthe lungs, the amount of gas confined around the lungs may increase overtime. As a result, to achieve the same pressure the gas volume controlhas to be re-calibrated, or the gas amount has to be re-adjusted, whichcan interrupt the normal ventilation cycles, or require manualre-adjustment of the device or the control settings.

In comparison, when the exterior pressure around the lungs is varied byapplying a gas pressure using a gas in fluid communication with a gaspump for varying the exterior pressure, the exterior pressure can beconveniently controlled continuously without interruption over a longperiod of time, by automatically adjusting the pump speed, optionallyaided with one or more proportioning valves that are configured toprovide more flexibility in the control of pressures and fluid flow asillustrated herein. Air leakage through the lungs will not affect thepressure control settings and thus will not cause interruption of theventilation process or require re-setting of the control parameters.

The analysis of lung physiology in vivo indicates that during ex vivoNPV, effective recruitment of lung parenchymal alveolar segmentsrequires that the TPG always be above zero, including above around 7cmH₂O. This latter value is analogous to the difference, at the end ofexpiration in vivo, between the airway pressure inside the lungs(atmospheric) and the intrathoracic pressure (around −7 cmH₂O); this TPGresists the elastic recoil of the alveoli and prevents alveolarcollapse. In general, herein, TPG during ex vivo NPV is the differencebetween the pressure inside the airways of the lung and the pressureinside the container housing the lung; it will be a positive value whenthe airway pressure is higher than the container pressure. A consequenceof the TPG always being greater than zero during ex vivo NPV may be acontinuous leakage of air into the container holding the lung, if, forinstance, the seal between the container and lid is not perfect, theseal between the endotracheal tube and an airway of the lung (e.g., thetrachea) is not perfect or if, for instance, the lungs have microscoperuptures, such as bronchopleural fistulae.

EXAMPLES Example I

The example apparatus and methods described in this disclosure (forexample, as shown in FIGS. 3-8 ) were used for EVLP and combinednegative and positive pressure ventilation (NPV/PPV) in a series ofexperiments on porcine lungs. For each experiment, a pair of lungs wasrapidly excised from a 40-50 kg pig following appropriate euthanasia andexsanguination. The lungs were mounted in the sealable container in theapparatus. An endotracheal tube was connected to the trachea of thelungs. The vasculature of the lungs was connected to a perfusion system,and perfusion was initiated. The perfusate comprised either an acellularalbumin solution, a mixture of whole blood and albumin solution, or amixture of concentrated red blood cells (pRBCs) and albumin solution.The composition of the albumin solution is presented in Table 1.

TABLE 1 Composition of albumin solution for lung perfusion Componentmmol/L g/4 L Glucose 10 7.20 NaCl 117 27.35 KCl 5.9 1.76 NaHCO₃ 25 8.40NaH₂PO₄ 1.2 0.66 CaCl₂ 1.12 0.66 MgCl₂ 0.512 0.42 sodium pyruvate 1 0.44bovine serum albumin 160 Adjust pH to 7.4

A computer controlled the proportioning valves and turbine in theapparatus using input from pressure sensors for P_(IT) and P_(AW), asdescribed for FIGS. 3-6 . The computer recorded data from the airwayflow sensor, as located in FIGS. 3-5 , but did not use this informationto control the apparatus. The computer was instructed to cause cycles ofinspiration and expiration by entering into the computer desiredset-points for: inspiratory time (t_(i)), expiratory time (t_(e)),end-inspiratory pressure (EIP) inside the sealed container,end-expiratory pressure (EEP) inside the sealed container, and aconstant positive airway pressure.

The lungs were perfused and ventilated for 12 hours. Data on lungmechanics as well as vascular function were collected continuously overthe 12 hour period. Perfusate samples were collected at regularintervals to measure dissolved gas content and inflammatory markerlevels.

FIGS. 10 to 17 present data derived from experiments on porcine lungsusing this NPV/PPV apparatus and method. In all of these figures, thepressures are all relative to atmospheric pressure (i.e., atmosphericpressure was 0 cmH₂O).

In addition, as a comparator, porcine lungs were subjected to the sameexperimental protocol mutatis mutandis with no NPV and with PPV providedby a standard ICU ventilator. FIG. 17 compares data obtained from theNPV/PPV protocol and this comparator.

The TPG could be accurately varied over time in response touser-inputted set-points to drive breathing ex vivo.

In FIG. 10 , the set-point for constant airway positive pressure was 10cmH₂O, the set-point for EIP was −10 cmH₂O, and the set-point for EEPwas 4 cmH₂O. FIG. 10 presents data for the measured container pressure(ITP), airway pressure (P_(aw)), and TPG during one inspiration andexpiration at these settings. At each time point, the TPG was calculatedas (P_(aw)−ITP). The TPG minimum was around 6 cmH₂O; the TPG maximum wasaround 20 cmH₂O.

FIG. 11 presents a pressure-volume loop from the same experiment as FIG.10 .

FIG. 12 provides the same type of data as FIG. 10 , but in thisexperiment the set-point for constant airway positive pressure was 4cmH₂O, the set-point for EIP was −30 cmH₂O, and the set-point for EEPwas 4 cmH₂O. The TPG minimum was around 0 cmH₂O; the TPG maximum wasaround 32 cmH₂O.

FIG. 13 presents a pressure-volume loop from the same experiment as FIG.12 .

FIGS. 14A to 14C show representative pressure profiles obtained in anexperiment, where sample porcine lungs were ventilated according to anembodiment of the present disclosure, in which a constant positiveairway pressure was directed into the airway of the lungs and the gaspressure (ITP) in a container surrounding the lungs was oscillatedbetween a higher pressure (slightly positive) and a lower pressure(negative). The set-points for the end expiratory pressure (EEP) and endinspiratory pressure (EIP) are indicated by different lines in FIG. 14A.FIG. 14A(b) is a magnified view of an initial time period in FIG.14A(a). The time period for FIG. 14A(a) was about 6 hours, and the timeperiod for FIG. 14A(b) was about 50 min. In both FIGS. 14A(a) and14A(b), the ITP was sampled every 10 seconds, such that although the ITPwas oscillating throughout the experiment to cause the lungs to breath,the apparent oscillations of ITP seen in FIG. 14A are not the actualoscillations of ITP. However, oscillation frequency shown in FIG. 14Awas inversely correlated with the actual times of expiration andinspiration.

For the experiment from which data is shown in FIGS. 14A to 14C, the EEPand EIP were set by a user at the various points in time, as indicatedin FIG. 14A. FIG. 14A shows that the observed pressure inside the sealedcontainer (ITP) changed over time in response to user-defined set-pointsfor EIP, EEP, t_(i),t_(e), and constant positive airway pressure.Set-points for EIP, EEP, t_(i), and t_(e) were serially altered duringthe experiment to challenge the apparatus. The CPAP set-point was onlychanged once: when around 15-20 min after the zero time point, the CPAPset-point was increased by the user to around 9 cmH₂O. In both FIGS.14A(a) and 14A(b), the ITP was sampled every 10 seconds, such thatalthough the actual ITP was oscillating throughout the experiment tocause the lungs to breath, the “observed ITP oscillations” seen in FIG.14A were not the actual oscillations of ITP. The frequency of the“observed ITP oscillations” shown in FIG. 14A was inversely correlatedwith the frequency of expiration and inspiration. FIG. 14A(b) is anexpanded view of an initial period of time, lasting around 50 min, fromFIG. 14A(a). The expanded view in FIG. 14A(b) shows the moretightly-spaced “observed ITP oscillations”, which were reflective of theshorter periods of the actual ITP oscillations due to lower t_(i) andt_(e) set-points in the initial portion of the profile. In addition,FIGS. 14A(a) and 14(b) show that when the user changed set-points, thesystem adjusted rapidly. FIG. 14B shows that throughout this experimentthe measured airway pressure (P_(AW)) remained fairly constant, perhapsshowing a gradual decrease over time, save for the initial portion ofthe profile where airway pressure rapidly increased in response to theaforementioned increase in the CPAP set-point. FIG. 14C shows themeasured TPG throughout the experiment, calculated as P_(AW)−ITP, whichwas kept between around 5 cmH₂O to around 26 cmH₂O for most of theexperiment.

FIG. 15 shows that the compliance (in mL/cmH₂O) of a porcine lungincreased over time (in 10 s) during perfusion and ventilation.Compliance was calculated as the volume of inspired air divided by thedifference between the TPG at the beginning and at the end of theinspiration.

FIG. 16 shows that the pulmonary vascular resistance (PVR, in dyn s/cm⁵)of a porcine lung decreased over time (in 10 s) during perfusion andventilation. As is known to one skilled in the art, when theconcentration of oxygen in the air of the alveoli decreases belownormal, the adjacent blood vessels constrict, thereby increasingvascular resistance. Therefore, the PVR can provide an indirect measureof alveolar recruitment and, more generally, provides a measure of thehealth of an ex vivo lung.

FIG. 17 shows that for the three different perfusates, edema formationduring EVLP of porcine lungs was less during ventilation in an NPV/PPVapparatus of the disclosure as compared to a standard PPV apparatus. Asis known to one skilled in the art, in normal human lungs in vivo, thereis a mean filtration pressure at the pulmonary capillary membrane whichcauses a continual flow of fluid from the pulmonary capillaries into theinterstitial spaces. This fluid is pumped back to the circulationthrough the pulmonary lymphatic system. The slight negative fluidpressure in the interstitial spaces keeps fluid from leaking into thealveoli. Any factor that causes this interstitial fluid pressure to riseinto the positive range can cause filling of the alveoli with freefluid. Therefore, reducing lung edema during ex vivo maintenance oflungs may be helpful.

Example II

Six human donor lungs were obtained by appropriate methods and mountedin the example apparatus shown in FIG. 3 and subjected to EVLP withNPV/PPV (N=6).

FIGS. 18 and 19 show representative flow-volume profiles for samplehuman lungs. Flow is presented in mL/s and volume is presented in mL.FIG. 18 shows a profile obtained during an initial ventilation periodwith an EIP set-point of −15 cmH₂O for preservation of the lungs.Ventilation was continued to allow recruitment of atelectatic alveoli.After the recruitment was completed, the lungs were ventilated at an EIPset-point of −18 cmH₂O for evaluation, and FIG. 19 shows a profileobtained during this evaluation period.

A flow-volume profile shows the relationship between inspiratory andexpiratory flow against the lung volume during maximal forcedinspiration and expiration. During expiration, flow was positive. Duringinspiration, flow was negative. The data points move clockwise with timein the profile.

FIGS. 18 and 19 demonstrate that the recruitment of alveoli duringventilation ex vivo in the NPV/PPV apparatus of the disclosure resultedin increased flow and vital capacity (i.e., the maximum volume expiredafter a maximum inspiration).

Example III

FIGS. 20 and 21 show representative pressure-volume profiles for samplehuman lungs ventilated ex vivo as in Example II. FIG. 20 shows a profileobtained in the initial ventilation during preservation, and FIG. 21shows a profile obtained during the later evaluation period.

FIGS. 20 and 21 also demonstrate the recruitment of alveoli duringventilation ex vivo in the NPV/PPV apparatus of the disclosure.

It was observed during experimentation, including that described inExamples I, II, and III, that the NPV/PPV apparatus and methods of thedisclosure had advantages relative to standard PPV apparatuses andmethods. The NPV/PPV apparatus and methods resulted in fasterrecruitment of the lung parenchyma (i.e., resolution of atelectasis),with a lower or equivalent TPG. There was less formation of bullae inthe donor lungs, with less broncho-pleural fistula formation andconsequently less air leak from the lungs. Broncho-pleural fistulae wereobserved as localized bubbling on the exterior surface of the lungs.Lower inflammatory marker expression in the perfusate was observed aswell. Lung edema occurred to a lesser degree.

It was also observed during experimentation that the NPV/PPV apparatusand methods of the disclosure, relative to a comparable NPV apparatusand method, resulted in lungs with superior physiological propertiesduring EVLP and ventilation ex vivo.

Example IV (Comparison)

FIG. 22 illustrates an example comparison apparatus 400 for negativepressure ventilation, which was tested with three porcine lungs (N=3).Apparatus 400 was used to apply a negative pressure within organ chamber410. In this example, apparatus 400 was not used to deliver positiveairway pressure in the airway of the lung.

In the tests, donor lungs 450 were placed inside a hard-shell sealedcontainer 410 in apparatus 400. The container 410 was partially filledwith a saline liquid 480. The lungs 450 floated on top of a flexibleplastic membrane 482, buoyed up by the liquid 480.

The tracheobronchial tree 452 of the lungs 450 was connected to aconduit 432 by an endotracheal tube 444. A conduit 462 connected theperfusion apparatus 460 to a pulmonary artery 454 of the lungs 450. Aconduit 464 connected the perfusion apparatus 460 to a pulmonary vein456 of the lungs 450.

The container 410 was sealed to the atmosphere by a lid 412. The conduit432 extended through the lid 412 via a port 414. The conduit 462extended through the lid 412 via a port 416. The conduit 464 extendedthrough the lid 412 via a port 418.

A conduit 430 connected the container 410 at an opening below the fluidlevel to an occlusive roller pump 402 (a COBE™ perfusion pump). Theinner space in container 410 was in fluid communication with conduit430. A conduit 434 connected the roller pump 402 to a fluid reservoir490.

Gas pressure inside the container 410 was lowered by pumping the liquid480 out of the container 410, into the conduit 430, through the rollerpump 402, into the conduit 434, and from thence into the reservoir 490.Gas pressure inside the container 410 was raised by pumping fluid in theopposite direction. By actuating the liquid 480 to and fro, the lungs450 were caused to breathe through the endotracheal tube 444 and theconduit 432, which was open to the atmosphere.

An HME filter 426 was coupled to the conduit 432 to avoid desiccation ofthe airways of the lungs 450.

A controller 470, which was a computer, controlled the speed anddirection of pumping by the roller pump 402. The control was implementedby optionally specifying a volume (e.g., tidal volume) to be removedfrom the container 410 or a target pressure (i.e., vacuum pressure) inthe container 410.

In the latter option, the pressure in the container 410 (the“intrathoracic” pressure or P_(IT)) was measured by a pressure sensor420. The pressure sensor 420 was an input into the controller 470.Unlike in Examples I-III and the apparatuses of FIGS. 2-6 , airwaypressure (P_(AW)) was not measured and was not a control input. The userspecified set-points for EIP, EEP, t_(i), and t_(e) instructed thecontroller 470 as to the desired ventilation cycles. The controller 470compared the pressure sensor 420 input to the user-defined set-points ateach given time to control the roller pump 402.

Endotracheal tube airflow (V) was measured by a flow sensor 428. Thisdata was recorded by the controller 470, but was not used to control theapparatus.

It was generally observed that the NPV apparatus 400 in comparativeExample IV was cumbersome. The NPV apparatus 400 did not allow forprecise and effective control of the TPG, as compared to the apparatusesof the disclosure tested in Examples I-III. In particular, highernegative pressures were required to cause effective inspiration,relative to the apparatuses in Examples I-III that combined NPV withPPV. In addition, altering the negative pressure by adjustingproportioning valves in the apparatuses of the disclosure was moreprecise and rapid than altering the negative pressure by adjusting thespeed and direction of the roller pump 402 in the apparatus ofcomparative Example IV.

FIG. 23 shows that the pressure (ITP, in cmH₂O) inside the sealedcontainer of the NPV apparatus 400 in FIG. 22 could be oscillated overtime.

The volume of the liquid 480 drawn from, or supplied to, the container410 by roller pump 402 was indicated by the step position of the rollerpump 402. Ideally, one might have expected that the volume of liquid 480withdrawn from and re-supplied to the container 410 during eachventilation cycle should be about the same, and the thus the median stepposition of the roller pump 402 should remain stable over time. However,as shown in FIG. 24 , experimental results indicated that the medianstep position of the roller pump 402 changed over time. Oscillations ofthe step position around the median step position resulted in liquid 480being moved in and out of the container 410 to cause the lung 450 toinspire and expire.

It is believed that some or all of the periods of steadily increasingmedian step position seen in FIG. 24 resulted from a slight leakage ofair into the container 410, which created a significant obstacle tolong-term ex vivo ventilation with the NPV apparatus 400.

It is believed that this air leakage was due to imperfections in theseal between the container 410 and the lid 412 (or its ports 414, 416,418), imperfections in the seal between the endotracheal tube 444 andthe lung airway 452, and/or from microscopic ruptures (e.g.,bronchopleural fistulae) in the wall of the lungs 450. Such ruptureswere, in fact, observed as inferred from the appearance of bubbles in alocalized portion of the surface of the lungs. It was observed that, ingeneral, excised lungs often have one or more such rupture.

The slight air leakage into container 410 had the apparent consequencethat, in each breathing cycle, the amount of liquid 480 removed from thecontainer 410 could be slightly greater than the amount of liquid 480returned to the container 410. As a result, the level of liquid 480 inthe container 410 could slowly decrease over time, which required thatthe breathing cycles be stopped, that the conduit 432 be clamped (so asto avoid collapse of the lungs), that the container 410 be opened to theatmosphere to add more liquid 480, and that the step position of thepump 402 be returned to zero. This is the procedure that was followedduring the times corresponding to each the two peaks at around 3.5 hr inFIG. 24 , where the steady rises in median step position correspond toobserved losses of the liquid 480 from the container 410 and the sharpdrops correspond to resetting the step position to zero after openingthe container and adding back more liquid 480.

In other portions of the experiment in FIGS. 22 to 24 , it was unclearwhy the median step position in FIG. 24 was rising and falling.

By contrast, in an NPV system of the present disclosure, no suchproblems with pumps or air leakage were encountered, whether with orwithout the application of PPV into the airway.

Example V

An apparatus and methods described in this disclosure (for example, asshown in FIGS. 3-8 ) were used for EVLP with positive pressureventilation (referred to as the “NPV/PPV-EVLP” platform) in a series ofexperiments on 16 porcine lungs and 3 human lungs. As previouslydescribed, the NPV/PPV-EVLP platform used a custom turbine drivenventilator to change the air pressure within the organ chamber. Theturbine and accompanying valve mechanism induced a negative pressurewithin the organ chamber and also delivered positive airway pressure,regulated with a positive-end-expiratory pressure (PEEP) valve.

As a comparator, an additional 16 porcine and 3 human lungs weresubjected to the same experimental protocol mutatis mutandis with onlyPPV (no NPV) provided by a standard ICU ventilator (referred to as the“PPV-EVLP” platform). The ventilator used was the SERVO-I™ provided byMaquet™ Critical Care AB of Solna, Sweden.

For each porcine lung experiment, a pair of lungs was rapidly excisedfrom a 37-47 kg female pig (approx. 2 to 3 months of age) followingappropriate euthanasia and exsanguination. The perfusate comprisedeither an acellular perfusate or a cellular perfusate. The acellularperfusate used was 2 L Krebs-Henseleit buffer with 8% bovine serumalbumin and the cellular perfusate used was 1.5 L Krebs-Henseleit bufferwith 8% bovine serum albumin+0.5 L packed red blood cells (pRBCs).

Each group of porcine lungs was split into two sub-groups, with eightlungs in each sub-group. The first sub-group (N=8) was perfused with thecellular perfusate with combined NPV/PPV; the second sub-group (N=8) wasperfused with the cellular perfusate with PPV; the third sub-group (N=8)was perfused with the acellular perfusate with combined NPV/PPV; and thefourth sub-group (N=8) was perfused with the acellular perfusate withPPV.

For each human lung experiment, a pair of lungs was rapidly excisedafter brain-stem death of the donor. The characteristics of each humandonor lung are presented in Table 2. The perfusate comprised a cellularperfusate (a solution of 1.5 L STEEN Solution™+0.5 L pRBCs was used).All tested human lungs were marginal and rejected for transplant. Thehuman lungs were split into two groups, with three lungs in each group.The first group (N=3) was perfused with the cellular perfusate withcombined NPV/PPV and the second group (N=3) was perfused with thecellular perfusate with PPV.

TABLE 2 Characteristics of human donor lungs Donor Donor Age WeightPO₂/FiO₂ Ventilation (Yrs) Sex (kg) (mmHg) Reason for Rejection PPV 72 M80 190 Age > 64; Poor oxygenation (<350 mmHg) PPV 54 M 80 270 High riskdonor; Poor oxygenation (<350 mmHg) PPV 16 F 64 80 Poor oxygenation &aspiration (<350 mmHg) NPV/PPV 80 F 80 98 MRSA pneumonia; Pooroxygenation (<350 mmHg) NPV/PPV 100 M 100 170 Size mismatch; Pooroxygenation (<350 mmHg) NPV/PPV 85 M 85 145 Emphysematic; ABO mismatch;Poor oxygenation (<350 mmHg)

The NPV/PPV-EVLP and PPV-EVLP platforms were primed with 2 liters of therespective experimental perfusate, 10,000 IU heparin, 500 mg ofmethylprednisolone, and 3.375 g of piperacillin/tazobactam. BothNPV/PPV-EVLP and PPV-EVLP platforms had a centrifugal pump (Medtronic™)that drove continuous flow of perfusate to the pulmonary artery (PA)from the reservoir (for NPV/PPV-EVLP platform, this was integrated inthe organ chamber, e.g. chamber 110 of FIG. 3). Perfusate initiallypassed through a M27 PH.I.S.I.0 adult arterial filter (Sorin GroupCanada Inc™), then a membrane de-oxygenator (Sorin PrimO2X™) and warmedwith a computer controlled heater (PolyScience™), prior to returning tothe lungs via the PA. Both platforms had a microcontroller with customsoftware that controlled the desired PA flow and monitored correspondingphysiologic parameters using a flow probe (BIO-Probe Transducer ModelTX40™ by Medtronic), pressure transducers (Edwards Lifesciences™), airpressure sensors, and an air flow meter. Data was collected at 10 sintervals. Both platforms utilized compressed medical air, a hypoxicsweep gas mix, (89% N₂, 8% CO₂, 3% O₂), to titrate pre-lung (PA)perfusate gas composition.

For the NPV/PPV-EVLP platform, a computer controlled the proportioningvalves and turbine in the apparatus using input from pressure sensorsfor P_(IT) and P_(AW), as described for FIGS. 3-6 . The computerrecorded data from the airway flow sensor, as located in FIGS. 3-5 , butdid not use this information to control the apparatus. The computer wasinstructed to cause cycles of inspiration and expiration by enteringinto the computer desired set-points for: inspiratory time (t_(i)),expiratory time (t_(e)), end-inspiratory pressure (EIP) inside thesealed container, end-expiratory pressure (EEP) inside the sealedcontainer, and a constant positive airway pressure.

For the PPV-EVLP platform, a Drager EVITA XL™ ventilator was used to setand control the ventilation parameters.

All lungs were perfused and ventilated for 12 hours. Data on lungmechanics as well as vascular function were collected continuously overthe 12 hour period. Perfusate samples were collected at regularintervals to measure dissolved gas content and inflammatory markerlevels.

To initiate each experiment in either the NPV/PPV-EVLP and PPV-EVLPplatforms, the pulmonary artery (PA) of each lung was cannulated, whilethe left atrium (LA) was left open, trachea was intubated with anendotracheal tube, and perfusion was initiated at 5% cardiac output (CO)and 20-25° C. (irrespective of experimental ventilation group).Anterograde perfusion was increased to 10% of predicted cardiac output(CO; CO =70 mL/kg/min) and perfusate was gradually warmed to 38° C. overa 60-minute period. The perfusate PA flow was increased by increments of10% of CO every 20-minutes of perfusion; thus, by T=1 (1 hour intoperfusion), a desired flow (preservation mode) of 30% CO was achieved.The initiation parameters used are shown in Table 3.

TABLE 3 Perfusion Initialization Parameters Perfusion 0 10 20 20-4040-50 60 (T = 1) Time (min.) Perfusion 20-30 25-30 32 32-34 34-3637.5-38 Temp (° C.) PA Flow 5 10 10 20 30 30 (% CO) Ventilation NoneNone Initiate Preser- Preser- Recruit- preser- vation vation ment vationmode mode phase mode Medical None None None None None Start Gas MixerLeft Atrial 0 0 0 0 0 0 Pressure (LAP; mmHg)

Experiments in the NPV/PPV-EVLP and PPV-EVLP platforms utilizedpressure-control ventilation and flow controlled perfusion. For bothplatforms, a preservation mode ventilation was initiated once theperfusate temperature reached 32° C. An evaluation ventilation mode(providing higher lung pressure and volume) was utilized for datacollection, thereby ensuring that the data collected on gas exchange andcompliance was done when the lungs were fully ventilated. Thepreservation and evaluative modes of ventilation and vascular pressureparameters are listed in Table 4.

TABLE 4 Preservation and Evaluation modes Preservation EvaluationVentilation Mode Ventilation Mode Temperature (° C.) 37.5 (Human) 37.5(Human) 38 (Porcine) 38 (Porcine) Pulmonary Artery Flow 30% CO 50% COVentilation Parameters Mode Volume Control Volume Control InspiratoryTidal Volume 6 10 (mL/kg) Frequency (bpm) 7-8 10-12 PEEP (cmH₂O) 7 5FiO₂ (%) 21 21 Pressure Parameters PAP (mmHg) <20 <20 LAP (mmHg) 0 0Medical gas mixer 89% N₂, 8% CO₂, 89% N₂, 8% CO₂, 3% O₂ 3% O₂ Medicalgas mixer (L/min) 35-55 35-50 titrated to PCO₂ (mmHg)

With the NPV/PPV-EVLP platform, to obtain the desired inspiratory tidalvolumes, the pleural pressure was varied between a negativeend-inspiratory-pressure (EIP) and an end-expiratory-pressure (EEP) thatwas slightly greater than airway pressure (Paw). The transpulmonary airpressure (TPG) was calculated: TPG=Paw−EIP. Evaluation was conductedserially every 2 hours, with upper peak airway pressure limit set to 25cmH₂O.

Sweep gas flow rate through the hollow fiber deoxygenator was titratedto maintain a physiological pH of 7.35-7.45 and PCO₂ (35-50 mmHg).Insulin (2.0 U/h) and glucose (1.0 g/h) were infused over the durationof EVLP.

For the first 3-hours of EVLP, the PEEP was maintained at 7 cmH₂O withinspiratory holds performed every 30 minutes for three consecutivebreaths (5-10 seconds/breath).

FIGS. 25A to 31C present data derived from experiments on lungs perfusedwith NPV/PPV-EVLP and lungs perfused with PPV-EVLP. In all of thesefigures, the pressures are all relative to atmospheric pressure (i.e.,atmospheric pressure was 0 cmH₂O).

Mean pulmonary artery pressure (mPAP), pulmonary vascular resistance(PVR), dynamic compliance (Cdyn), peak airway pressure (PAWP), and ratioof arterial partial pressure of oxygen to the oxygen fraction ininspired air (PO₂/FiO₂ or P/F ratio) were measured during the evaluativetime points.

FIGS. 25A, 25B, and 25C illustrate results of measurements of lungoxygenation (i.e. the PO₂/FiO₂ ratio or the P/F ratio, measured in mmHg)of the perfused lungs over time for lungs perfused with combined NPV/PPVand lungs perfused with PPV. In particular, FIG. 25A illustrates resultsof the porcine lungs perfused with acellular perfusate; FIG. 25Billustrates results of the porcine lungs perfused with cellularperfusate; and FIG. 25C illustrates results of the human lungs perfusedwith cellular perfusate. The results illustrate that lung oxygenationremained at an acceptable level of more than 400 mmHg for both porcineand human lungs perfused with combined NPV/PPV or PPV. There was nostatistically significant (i.e. p>0.05) difference in lung oxygenationbetween lungs perfused with either type of perfusate and between lungsperfused with either ventilation platform. However, only lungs perfusedwith the cellular perfusate demonstrated a statistically significantimprovement in oxygenation over time.

FIGS. 26A, 26B, and 26C illustrate results of measurements of meanpulmonary arterial pressure (mPAP; measured in mmHg) of the perfusedlungs over time for lungs perfused with combined NPV/PPV and lungsperfused with PPV. In particular, FIG. 26A illustrates results of theporcine lungs perfused with acellular perfusate; FIG. 26B illustratesresults of the porcine lungs perfused with cellular perfusate; and FIG.26C illustrates results of the human lungs perfused with cellularperfusate. Similarly, FIGS. 27A, 27B, and 27C illustrate results ofmeasurements of pulmonary vascular resistance (PVR; measured in dyns/cm⁵) of the perfused lungs over time for lungs perfused with combinedNPV/PPV and lungs perfused with PPV. In particular, FIG. 27A illustratesresults of the porcine lungs perfused with acellular perfusate; FIG. 27Billustrates results of the porcine lungs perfused with cellularperfusate; and FIG. 27C illustrates results of the human lungs perfusedwith cellular perfusate. As shown in FIGS. 26A-27C, all the porcinelungs demonstrated a statistically significant decline in mPAP and PVR.However, there was no statistically significant difference in resultsbetween lungs perfused with combined NPV/PPV and lungs perfused withPPV. Further, there was no statistically significant change over time inmPAP and PVR for human lungs perfused with either PPV or combinedNPV/PPV (see FIGS. 26C and 27C respectively).

FIGS. 28A, 28B, and 28C illustrate results of measurements of peakairway pressure (P_(AWP); measured in cmH₂O) of the perfused lungs overtime for lungs perfused with combined NPV/PPV and lungs perfused withPPV. In particular, FIG. 28A illustrates results of the porcine lungsperfused with acellular perfusate; FIG. 28B illustrates results of theporcine lungs perfused with cellular perfusate; and FIG. 28C illustratesresults of the human lungs perfused with cellular perfusate. In allcases (porcine-cellular, porcine-acellular, and human-cellular), therewas no statistically significant difference in P_(AWP) between lungsperfused with combined NPV/PPV and lungs perfused with PPV. However, asshown in FIG. 28B, P_(AWP) showed a statistically significant decreaseover time for porcine lungs perfused with cellular perfusate andcombined NPV/PPV.

FIGS. 29A, 29B, and 29C illustrate results of measurements of dynamiccompliance (C_(dyn); measured in mL/cmH₂O) of the perfused lungs overtime for lungs perfused with combined NPV/PPV and lungs perfused withPPV. In particular, FIG. 29A illustrates results of the porcine lungsperfused with acellular perfusate; FIG. 29B illustrates results of theporcine lungs perfused with cellular perfusate; and FIG. 29C illustratesresults of the human lungs perfused with cellular perfusate. Porcinelungs perfused with the cellular perfusate demonstrated a statisticallysignificant improvement in compliance over time (FIG. 29B). For example,as shown in FIG. 29B at T=11, the C_(dyn) for lungs perfused withcombined NPV/PPV was 29.3±1.6 mL/cmH₂O and the C_(dyn) for lungsperfused with PPV was 24.5±1.5 mL/cmH₂O. However, the same trend was notobserved in porcine lungs perfused with acellular perfusate (FIG. 29C),irrespective of the ventilation platform. In contrast, perfused humanlungs demonstrated statistically significantly improving compliance overtime only when perfused with combined NPV/PPV.

Pro-inflammatory cytokine profiles (including tumor necrosis factor-α(TNFα), interleukin-6 (IL-6), and interleukin-8 (IL-8)) were analyzedusing enzyme-linked immunosorbent assay (ELISA) kits provided by R&DSystems™.

FIGS. 30A-30F illustrate results of measurements of inflammatorycytokine of the perfused porcine lungs over time for lungs perfused withcombined NPV/PPV and lungs perfused with PPV. FIGS. 30A and 30B show theconcentration, in pg/mL, of tumor necrosis factor alpha (TNFα) for lungsperfused with the cellular perfusate and for lungs perfused with theacellular perfusate, respectively; FIGS. 30C and 30D show theconcentration, in pg/mL, of Interleukin 6 (IL-6) for lungs perfused withthe cellular perfusate and for lungs perfused with the acellularperfusate, respectively; and FIGS. 30E and 30F show the concentration,in pg/mL, of Interleukin 8 (IL-8) for lungs perfused with the cellularperfusate and for lungs perfused with the acellular perfusate,respectively. As shown in FIGS. 30A-30F, a statistically significantlylower pro-inflammatory cytokine production in porcine lungs perfusedwith combined NPV/PPV irrespective of the perfusate used was observed.

Similarly, FIGS. 31A-31C illustrate results of measurements ofinflammatory cytokine of the perfused human lungs over time for lungsperfused with combined NPV/PPV and lungs perfused with PPV. FIG. 31Ashows the concentration, in pg/mL, of tumor necrosis factor alpha(TNFα); FIG. 31B shows the concentration, in pg/mL, of Interleukin 6(IL-6); and FIG. 31C shows the concentration, in pg/mL, of Interleukin 8(IL-8). As shown in FIGS. 31A and 31B, at all times there astatistically significantly lower TNFα and IL-6 production in humanlungs perfused with combined NPV/PPV than those perfused with PPV wasobserved. However, as shown in FIG. 31C, observed was a statisticallysignificantly lower IL-8 production in human lungs perfused withcombined NPV/PPV than in human lungs perfused with PPV only at T=3 andT=11.

Bullae formation during EVLP was counted at T=12. Human lungs perfusedwith PPV and human lungs perfused with combined NPV/PPV did not developbullae. For porcine lungs, bullae were counted at 21.4% for lungsperfused with combined NPV/PPV, and 63.8% for lungs perfused with PPV.Accordingly, among perfused porcine lungs, there was 42% lower incidenceof bullae formation in lungs perfused with combined NPV/PPV in contrastto those perfused with PPV. A lower bullae formation for porcine lungsperfused with combined NPV/PPV compared with lungs perfused with PPV wasobserved.

Lungs were also weighed before and after EVLP to calculate the globaledema as a weight gain percentage. At T=12, there was less edemaformation (i.e. weight gain) in porcine lungs perfused with combinedNPV/PPV relative to those perfused with PPV for both perfusates (Forcellular perfusate, contrast 20.1±4.1% of edema formation for combinedNPV/PPV with 39.0±6.6% of edema formation for PPV; and for acellularperfusate, contrast 40.4±5.3% of edema formation for combined NPV/PPVwith 88.1±11.0% of edema formation for PPV).

For human lungs, a drying effect (i.e. a weight reduction) was observedfor lungs perfused with combined NPV/PPV in contrast with an edemaformation (i.e. weight gain) for lungs perfused with PPV (contrast−8.0±2.1% for combined NPV/PPV with +39.4±5.7% of edema formation forPPV). The reduction in lung weight from baseline suggests that perfusionof human lungs with combined NPV/PPV may help reverse the state of lungedema that had occurred in a donor lung. In particular, since the humanlungs used in Example V were fragile/marginal (as they were obtainedfrom rejected donors and had varying degree of lung injury), perfusionwith combined NPV/PPV may transform a fragile/marginal into a suitablelung for donation.

Human peripheral lung tissue biopsies were collected at the end of EVLP(T=12). Biopsies were fixed in 10% buffered formalin for 24 hours,embedded in paraffin, sectioned at 5-μm thickness, stained byhematoxylin-eosin (H&E), and examined for pathological changes withlight microscopy. Representative photomicrographs of human lung tissuewere obtained after T=12 hours of EVLP (not shown). A blinded pulmonarypathologist graded the lung sections in a randomized fashion to assessthe histopathological grading of acute lung injury. Thehistopathological grading of acute lung injury was calculated inaccordance with the methods set out in Mehaffey J H, Charles E J, SharmaA K, et al, “Airway pressure release ventilation during ex vivo lungperfusion attenuates injury”, J Thorac Cardiovasc Surg 2017; 153:197-204 and Tane S, Noda K, Shigemura N, “Ex Vivo Lung Perfusion: A KeyTool for Translational Science in the Lungs”, Chest 2017.

Overall, a lower acute lunge injury was observed by histopathology forhuman lungs perfused with combined NPV/PPV in comparison with lungsperfused with PPV. For example, the interstitial edema histologicalscore for the lung perfused with combined NPV/PPV was determined to be1.5, whereas for the lung perfused with PPV, the histological score wasdetermined to be 2.7. Further, the alveolar inflammation histologicalscore for the lung perfused with combined NPV/PPV was determined to be1.5, whereas for the lung perfused with PPV, the histological score wasdetermined to be 2.7. Further, the amount of neutrophilic infiltratesfor the lung perfused with combined NPV/PPV was statisticallysignificantly lower (observed to have an infiltration density of 6.3)than the amount of neutrophilic infiltrates for the lung perfused withPPV (observed to have an infiltration density of 14.8). Further, theinterstitial inflammation histological score for the lung perfused withcombined NPV/PPV was determined to be 1.2, whereas for the lung perfusedwith PPV, the histological score was determined to be 1.7 (which was notstatistically significant; p>0.05). Further, the hemorrhage histologicalscore for the lung perfused with combined NPV/PPV was determined to be0.0, whereas for the lung perfused with PPV, the histological score wasdetermined to be 0.7 (which was also not statistically significant;p>0.05). Further, the perivascular neutrophil infiltration density wasobserved to be 0.6 for the lung perfused with combined NPV/PPV, and 1.6for the lung perfused with PPV.

The results obtained from Example V (outlined above) suggest that lungsperfused with the NPV/PPV-EVLP platform may suffer lower rates ofventilator induced lung injury (VILI). Lungs perfused with combinedNPV/PPV were observed to have stable and acceptable physiologicparameters over 12 hours of EVLP. The physiologic parameters for lungsperfused with combined NPV/PPV were observed to be similar to those oflungs perfused with PPV (see, for example, FIGS. 25A-27C).

Further, lungs perfused with combined NPV/PPV were observed to havesuperior results in comparison with lungs perfused with PPV. Forexample, lungs perfused with combined NPV/PPV were observed to have adecreased production of pro-inflammatory cytokines compared to lungsperfused with PPV (FIGS. 30A-31C), a decreased incidence of bullaeformation compared to lungs perfused with PPV, and decreased lung edemain both porcine and human lungs compared to lungs perfused with PPV.Further, human lungs perfused with combined NPV/PPV were observed tohave a decreased histopathologic finding of acute lung injury comparedto lungs perfused with PPV. Accordingly, while the physiologicparameters over 12 hours of EVLP were similar for lungs perfused withcombined NPV/PPV to lungs perfused with PPV, the lungs perfused with PPVwere observed to have a sub-clinical deterioration in quality.

CONCLUDING REMARKS

Selected Embodiments of the present invention may be used in a varietyof fields and applications. For example, they may have applications intransplantation surgery and research.

Other features, modifications, and applications of the embodimentsdescribed here may be understood by those skilled in the art in view ofthe disclosure herein.

It will be understood that any range of values herein is intended tospecifically include any intermediate value or sub-range within thegiven range, and all such intermediate values and sub-ranges areindividually and specifically disclosed.

The word “include” or its variations such as “includes” or “including”will be understood to imply the inclusion of a stated integer or groupsof integers but not the exclusion of any other integer or group ofintegers.

It will also be understood that the word “a” or “an” is intended to mean“one or more” or “at least one”, and any singular form is intended toinclude plurals herein.

It will be further understood that the term “comprise”, including anyvariation thereof, is intended to be open-ended and means “include, butnot limited to,” unless otherwise specifically indicated to thecontrary.

When a list of items is given herein with an “or” before the last item,any one of the listed items or any suitable combination of two or moreof the listed items may be selected and used.

Of course, the above described embodiments of the present disclosure areintended to be illustrative only and in no way limiting. The describedembodiments are susceptible to many modifications of form, arrangementof parts, details and order of operation. The invention, rather, isintended to encompass all such modification within its scope, as definedby the claims.

What is claimed is:
 1. A system for ventilating a lung, comprising: agas pump comprising an exhaust side and an intake side; a sealed chamberfor enclosing the lung therein, and configured to apply a first pressure(P1) to an airway of the lung and apply a second pressure (P2) to anexterior surface of the lung enclosed therein; and a plurality ofconduits and a plurality of valves, the conduits and valves connectingthe gas pump to the sealed chamber for selectively regulating P1 and P2,wherein the proportional valves comprise a first proportional valveconnected to the exhaust side of the gas pump, a second proportionalvalve connected to the intake side of the gas pump, and a thirdproportional valve connected to the first proportional valve, andwherein the conduits comprise a first conduit extending through thesealed chamber and connected to the third proportional valve, forconnecting an airway of the lung to the gas pump through the thirdproportional valve and the first proportional valve to supply thepressure applied to the airway of the lung, a second conduit connectingthe second proportional valve to the sealed chamber for supplying thepressure applied to the exterior surface of the lung, and a thirdconduit connecting the second conduit to the first proportional valve,such that P1 is regulated by the first proportional valve and the thirdproportional valve, P2 is regulated by the first proportional valve andthe second proportional valve, and P1 and P2 are independentlyregulatable by controlling the first, second and third proportionalvalves.
 2. The system of claim 1, comprising a controller forcontrolling operations of the gas pump and the plurality of valves. 3.The system of claim 1, wherein the gas pump comprises a regenerativevacuum pump.
 4. The system of claim 3, wherein the regenerative vacuumpump is a regenerative turbine.
 5. The system of claim 1, wherein thegas pump comprises a single pump.
 6. The system of claim 1, wherein thegas pump comprises a single turbine.
 7. The system of claim 1, whereinthe sealed chamber has a rigid internal wall defining a constantinternal volume.
 8. The system of claim 1, comprising pressure sensorsfor detecting pressures at different locations in the plurality ofconduits and the sealed chamber.
 9. The system of claim 1, comprisingflow sensors for detecting fluid flow rates at different locations inthe plurality of conduits.
 10. A lung ventilator comprising: a containercomprising a sealable chamber for housing a lung therein; a pumpcomprising an intake side and an exhaust side; a plurality of conduitsconnecting the pump to the sealable chamber for applying a firstpressure (P1) to an airway of the lung and a second pressure (P2) to anexterior surface of the lung; and a control system comprising acontroller, pressure sensors, flow sensors, and flow regulating valves,for controlling operation of the pump and regulating the pressuresapplied to the airway and the exterior surface of the lung, wherein theflow regulating valves comprise a plurality of proportional valvesconfigured to regulate P1 using an exhaust pressure at the exhaust sideof the pump, to regulate P2 using both the exhaust pressure and anintake pressure at the intake side of the pump, such that P1 and P2 areindependently controllable by adjusting proportional valves.
 11. Theapparatus of claim 10, wherein the pump comprises a regenerative vacuumpump.
 12. The apparatus of claim 11, wherein the pump is a regenerativeturbine.
 13. The apparatus of claim 11, wherein the pump is a singleturbine.
 14. A method of ventilating a lung, comprising: applying afirst pressure (P1) to an airway of the lung, wherein P1 is above anatmospheric pressure; applying a second pressure (P2) to an exteriorsurface of the lung; and varying P2 to change a pressure differential(PD) so as to cause the lung to breathe, wherein PD=P1−P2.